1. Field of the Invention
The present invention comprises an apparatus and method for making a gradient gel, and more particularly, the present invention relates to an apparatus and method of using a movable dispensing device to form a uniform linear gradient across a wide gel that provides more than twenty sample lanes so that more than forty samples can be analyzed simultaneously with a conventional dual-gel electrophoretic chamber.
2. Background Art
Sixty to seventy five percent of the cholesterol in blood is associated with low density lipoproteins (xe2x80x9cLDLxe2x80x9d) which consist of a non-homogeneous mixture of spherical particles ranging widely in particle size (23-28 nm), buoyant density and chemical composition. Using a non-denaturing 2-16% polyacrylamide gradient gel electrophoresis, researchers have noted that individuals with a high-risk lipid profile were most likely to have primarily small, dense LDL particles, as discussed in the paper xe2x80x9cGenetic control of low density lipoprotein subclasses,xe2x80x9d Austin et al., Lancet 2: 592-595(3)(1986). In a case-control study of men and women with documented myocardial infarction (MI) published in the paper xe2x80x9cLow-density lipoprotein subclass patterns and risk of myocardial infarction,xe2x80x9d Austin et al., J. Amer. Med. Assoc. 260: 1917-1921(4) (1988), it was reported that LDL phenotype B, the LDL subclass pattern characterized by a preponderance of small dense LDL particles, was associated with a 3-fold increased risk of MI. This association remained significant after adjustment for age, sex and relative weight. It has also been suggested that there may be a major genetic determinant for this LDL phenotype as in the paper xe2x80x9cInheritance of Low-density lipoprotein subclass patterns: results of complex segregation analysis,xe2x80x9d Austin et al., Am J Hum Genet 73: 838-876 (5) (1988). Whether or not the relationship between LDL phenotype and CAD is independent of other risk factors such LDLc, HDLc or TRIG is still unclear.
High density lipoproteins (HDL) are responsible for the reverse transport of cholesterol from peripheral tissues back to the liver. Data from the paper xe2x80x9cAltered particle size distribution of apoA-I-containing lipoproteins in subjects with coronary artery disease,xe2x80x9d Cheung et al., J. Lipid Res. 32: 383-397 (1991), and the paper xe2x80x9cCharacterization of human high density lipoproteins by gradient gel electrophoresis,xe2x80x9d Johansson et al, Biochim Biophys Acta 665: 708-719 (1991), would suggest that patients with documented CAD may have altered HDL particle size distribution when compared to that observed in non-CAD controls. In these studies, the heterogeneity of plasma HDL was assessed using a non-denaturing 4-30% polyacrylamide gradient gel first described in the paper xe2x80x9cCharacterization of human high density lipoproteins by gradient gel electrophoresis,xe2x80x9d Blanche et al., Biochim Biophys Acta 665: 708-719 (1981).
A major impediment to large prospective studies of lipoprotein particle size distribution has been the unavailability of an efficient and reproducible method that can allow the determination of particle diameters for cholesterol-rich lipoproteins. This is mainly because high quality pre-cast gradient gels used in the earlier studies are no longer available commercially. The paper xe2x80x9cProduction of polyacrylamide gradient gels for the electrophoretic resolution of lipoproteins,xe2x80x9d Rainwater et al., J. Lipid. Res. 33: 1876-1881 (1992), has reported a procedure for the preparation of a 4-30% gradient gel which provides estimates of HDL particle size comparable to those obtained with the PAA 4/30 gel (Pharmacia). In this gradient, however, LDL and larger lipoprotein particles tend to accumulate at the top of the gel, prohibiting the determination of particle size of these lipoproteins. A custom-made 2-16% gradient gel was also described by these investigators for the determination of LDL particle size in the paper xe2x80x9cEffects of diabetes on lipoprotein size,xe2x80x9d Singh et al., Arterioscl. Thromb. Vasc. Biol. 15: 1805-1811 (1995). Except for Gambert et al., who used lipid staining to visualize the LDL band as disclosed in the paper xe2x80x9cHuman low density lipoprotein fractions separated by gradient gel electrophoresis: Composition, distribution and alterations induced by cholesteryl ester transfer protein,xe2x80x9d J. Lipid. Res. 31: 1199-1210 (1990), most investigators used Coomassie to stain the gels for protein after the electrophoresis. The use of a protein stain typically requires extensive staining and de-staining procedures for the gels after electrophoresis and special handling of the gels during these steps to maintain gel size and shape before scanning. Furthermore, by using a protein stain, many protein bands other than those corresponding to plasma lipoproteins are visible from the electrophoresis of whole plasma.
It is very difficult to make high quality of gradient gels for medical studies and clinic use. In the casting of the typical gradient gels, as shown in Rainwater et al. paper, the polyacrylamide solutions are commonly allowed to flow into a gel chamber from a stationary dispensing tip which is typically placed at the center of the gel. However, as the polyacrylamide solution flows from the dispensing tip to the sides of the plate, a secondary gradient is formed across the width of the gel resulting in lower gel concentrations toward the edges because of the diffusion of the solution. In order to reduce this diffusion effect, only narrow gels with 6-8 lanes across have been available to-date although a typical gel chamber is capable of having gels with up to 20 or more lanes. Moreover, uneven gradients and disturbances in the process of gel making due to the diffusion still exist even in the narrow gels.
Definitions
A number abbreviations used in this application for some frequently used technical terms are defined as the following:
The term xe2x80x9cS-GGExe2x80x9d as used herein shall refer to a segmental gradient gel electrophoresis.
The term xe2x80x9cS-GGE 2.8/8.30xe2x80x9d as used herein shall refer to a 2.8/8.30 segmental gradient gel electrophoresis with a 2-8% gradient stacked above an 8-30% gradient.
The term xe2x80x9cLIPOPROTEINxe2x80x9d as used herein shall refer to a class of plasma proteins that are complexed to lipids.
The term xe2x80x9cTRIGxe2x80x9d as used herein shall refer to triglycerides.
The term xe2x80x9cCHOLxe2x80x9d as used herein shall refer to cholesterol.
The term xe2x80x9cLDLxe2x80x9d as used herein shall refer to low density lipoproteins.
The term xe2x80x9cHDLxe2x80x9d as used herein shall refer to high density lipoproteins.
The term xe2x80x9cLp(a)xe2x80x9d as used herein shall refer to lipoprotein(a) which consist of one LDL particle complexed to one apo(a) particle.
The term xe2x80x9cLpBxe2x80x9d as used herein shall refer to apob-containing lipoproteins.
The term xe2x80x9cLDLcxe2x80x9d as used herein shall refer to LDL-cholesterol.
The term xe2x80x9cHDLcxe2x80x9d as used herein shall refer to HDL-cholesterol.
The term xe2x80x9cLpA-Ixe2x80x9d as used herein shall refer to apoA-I containing lipoproteins.
The term xe2x80x9cLpA-I/A-IIxe2x80x9d as used herein shall refer to lipoproteins containing both apoA-I and apoA-II.
The present invention provides a new apparatus and method for making a uniform gel including a uniform, continuous gradient gel in many lanes occupying up to the capacity of a gel chamber. Moreover, the present invention can be practiced to produce a segmental gradient gel that would provide optimal conditions for the simultaneous characterization of LDL, Lp(a) and remnant lipoproteins (2-8% gradient) and HDL subclasses (8-30% gradient) from whole plasma. Additionally, the present invention allows the bands corresponding to all of the major lipid-carrying particles to be visualized without any handling of the gel. The present invention can also be practiced to make several gradient gels simultaneously. In sum, the present invention offers a new, better, and efficient gel making apparatus and method.
The present invention in one embodiment is a gel-making system that has a reservoir for holding a solution. The reservoir is connected to a movable arm through a tubing. The tubing has two ends: one end is in fluid communication with the reservoir; and the other, an open end, is received by the movable arm. A gel holder having an internal gel chamber is placed underneath the movable arm for receiving the solution. In operation, the movement of the movable arm causes the open end of the tubing to move along with it and the open end of the tubing delivers the solution in motion to the internal gel chamber to form the gel.
In order to make a gradient gel, normally two solutions with different concentrations are used. Accordingly, one embodiment of the present invention employs a gradient maker that consists of a reservoir having a first container and a second container. The first container holds a first solution and the second container holds a second solution. A channel connects the first container and the second container with an outlet connected to the second container and in communication with the channel so that a fluid of the first solution and the second solution is formed at the outlet. A tubing having a first end and a second end connects to the outlet with the first end. The second end of the tubing is received by a movable arm and moves along with the movable arm. A gel holder with an internal gel chamber is placed underneath the movable arm for receiving the fluid, where the chamber has a longitudinal axis. The movable arm moves back and forth along the longitudinal axis so that the second end of the tubing delivers the fluid in motion in the gel chamber to form the gradient gel. In a linear gradient gel the bottom of the gel has a higher concentration and the top of the gel has a lower concentration.
A linear gradient gel can be formed from more than two solutions. In another embodiment of the present invention, a reservoir has a plurality of containers holding a plurality of solutions. Each container holds one solution and communicates with at least one neighboring container. An outlet is connected to at least one container to communicate with the containers so that a fluid of at least two solutions from the plurality of solutions is formed at the outlet. A tubing, having a first end and a second end, is connected to (and is in fluid communication with) the outlet through the first end. A movable arm receives the second end of the tubing and causes the second end of the tubing to move along with it. A gel holder with an internal gel chamber is placed underneath the movable arm for receiving the fluid, where the chamber has a longitudinal axis. The movable arm moves back and forth along the longitudinal axis of the chamber so that the fluid is transferred from the first end to the second end of the tubing and then is delivered in the chamber by the second end of the tubing in motion to form the gradient gel.
In gel making, approximately two hours are required for the gel solution to polymerize and form a solid matrix. One advantage of the present invention is that several highly uniform gradient gels can be made simultaneously. In one embodiment of the present invention, a plurality of reservoirs are utilized. Each of them has a first container and a second container, where the first container holds a first solution and the second container holds a second solution. A channel connects the first container and the second container. Moreover, an outlet is connected to the second container and communicates with the channel. Consequently, a fluid of the first solution and the second solution is formed at the outlet of this particular reservoir. Thus, a plurality of fluids are formed at the plurality of the outlets of the plurality of the reservoirs. Furthermore, a plurality of tubings, each having a first end and a second end, connect in a one to one relationship to the plurality of reservoirs through the connection of the second end of a tubing to the outlet of a reservoir. A movable arm carries at least a plurality of the second ends of the plurality of the tubings. And a plurality of gel holders are positioned in parallel thereby defining a longitudinal axis. Each of the gel holders has an internal gel chamber for receiving the fluid from one of the plurality of the second ends. When the movable arm moves back and forth along the longitudinal axis, each second end of the tubings delivers one of the fluids in motion into one gel chamber to form one gradient gel. As a result, a plurality of the gradient gels are produced.
In order to make an uniform gradient gel, the movable arm moves at a substantially constant rate of motion. While other mechanisms may be used, one embodiment of the present invention uses a motor to drive the movable arm. One advantage of using the motor driving mechanism is that by adjusting the speed of the motor, the rate of motion of the movable arm can be selected. In order to ensure that the same gel concentration is present across the width of the gel, the rate of motion of the moveable arm is adjusted and set according to the width of the gel and the rate of flow of the solution from the reservoir into the gel chamber. For instance, for a wider gel, the rate of motion should be increased to cover a greater distance in the same interval of time. Also, the height of the reservoir relative to the gel chamber can affect the flow rate, which can be readily taken into account when setting the rate of motion of the movable arm. According to the preferred embodiment, the movable arm has an internally threaded bore and the motor controls the motion of the movable arm through a shaft. The shaft has an elongated body with an external thread on the elongated body. The shaft has a longitudinal axis and is rotatable around its longitudinal axis. The shaft can rotate around the longitudinal axis either in clockwise direction or counter-clockwise direction, and the direction of rotation of the shaft is changeable from clockwise to counter clockwise, or vice versa. The external thread on the elongated body is adapted to mate with the movable arm through the internally threaded bore. The shaft operatively connects to the motor. Thus, when the motor causes the shaft to rotate around its longitudinal axis, the movable arm moves along the longitudinal axis because the mating mechanism of the external thread of the shaft with the internally threaded bore of the movable arm. Because the shaft can rotate around the longitudinal axis either in clockwise direction or counter-clockwise direction, the movable arm is able to move along the longitudinal axis both forward and backward. A switching mechanism may be used to control the direction of the rotation of the shaft.
The movable arm in turn receives an open end of the tubing, which connects with the reservoir at the other end, the open end moving with the movable arm. To do so, the movable arm has means for holding at least a portion of the tubing proximate to the open end of the tubing so that at least the open end of the tubing moves along with the movable arm. In one embodiment of the present invention, the holding means is an opening sized to allow the open end of the tubing to pass through but hold at least the portion of the tubing proximate to the open end of the tubing therein. The movable arm may have several openings at different locations to allow several gels to be made simultaneously, or to give a user freedom to set up the user""s devices. The openings can also have different sizes to accommodate tubings with different sizes. Alternatively, other holding means, including a clamping device normally used in laboratories such as a clamp, can be used to associate the tubing with the moveable arm.
The tubing is made from a flexible material so that it can move along with the movable arm easily without impeding the flow of the fluid within the tubing. Many materials can be used. In one embodiment of the present invention, tubings made from Manosie silicone rubber by VWR Scientific Products Corporation, located at Willard, Ohio are used. In use, the tubing transfers fluid from the reservoir and delivers the fluid in motion at a substantially constant rate of flow. The tubing may deliver the fluid through a dispensing tip. Or, the fluid can be delivered simply through an open end of the tubing.
Solutions to make a linear gradient gel normally are polyacrylamide solutions. These solutions can be identified as high concentration polyacrylamide solution, medium concentration polyacrylamide solution and low concentration polyacrylamide solution. For a preferred embodiment of the present invention, a solution according to a mixing ratio of a 2.3 g acrylamide and 0.1 g N,Nxe2x80x2-methylene-bis-acrylamide in 100 ml of borate buffer is regarded as a low concentration polyacrylarnide solution (2.4%), a solution according to a mixing ratio of a 10.24 g acrylamide and 0.43 g N,Nxe2x80x2-methylenebis-acrylamide in 100 ml of borate buffer is regarded as a medium concentration polyacrylamide solution (10.67%), and a solution according to a mixing ratio of a 38.4 g of acrylamide and 1.6 g N,Nxe2x80x2-methylene-bis-acrylamide in 100 ml of borate buffer is regarded as a high concentration polyacrylamide solution (40%).
These solutions have been used successfully in the present invention to produce high quality segmental linear gradient gels, namely, a 2-8% continuous linear gradient gel and a 8-30% continuous linear gradient gel. In doing so in one embodiment of the present invention, a commercial gradient maker with a container A and a container B, such as a Hoefer GS 100, purchased from Hoefer Scientific Instr., San Francisco, Calif. is loaded with solutions. Container A and container B connect to each other through a channel. An outlet connects to the container B and communicates with the channel so that a mixed fluid of the first solution and the second solution is formed at the outlet. For this embodiment, the higher concentration of solution is always in container B. To make a 8-30% linear gradient gel, a high concentration of polyacrylamide is in container B and a medium concentration of polyacrylamide is in container A. A tubing transfers the mixed fluid of the high concentration of polyacrylamide and the medium concentration of polyacrylamide from the outlet to a dispensing tip. The dispensing tip can be a separate device. Or an open end of the tubing can function as the dispensing tip. A movable arm receives the dispensing tip. A gel holder having an internal gel chamber with a longitudinal axis is placed underneath the dispensing tip. The motion of the movable arm along the longitudinal axis causes the dispensing tip to move along with movable arm and the dispensing tip delivers the fluid in motion in the gel chamber. Because the motion of the movable arm can be controlled at a substantially constant rate, the fluid can be evenly delivered throughout the gel chamber. Moreover, because the movable arm is capable of traveling the length of the gel chamber, the solution is delivered directly to the gel chamber xe2x80x9con-the-spot.xe2x80x9d Thus, a secondary gradient resulting from the diffusion of the solution from the dispensing tip to the edge of the gel is not formed. After a proper curing period, a uniform, high quality 8-30% gradient gel is produced. Since in the segmental linear gradient gel a second gel (2-8% segment) must be poured on top of the first gel (8-30% segment), the volume of the solutions is controlled so that the 8-30% gradient gel occupies half space of the gel chamber. As people skilled in the art appreciate, the exact volumes to be placed in the reservoir can be calculated from the dimension of the gel chamber before hand. Furthermore, the entire gel making process according to the present invention can be automated by placing the right volumes of the solutions in the reservoir. The gel is poured to completion until the reservoir is emptied.
The containers A and B are subsequently filled with a medium concentration of polyacrylamide (in container B) and a low concentration of polyacrylamide (in container A) to make a 2-8% gradient gel. The above process is then repeated. And a uniform 2-8% gradient gel is formed on top of the 8-30% gradient gel. As a result, a segmental linear gel consisting of two continuous linear gradients is formed, which can then be used for the simultaneous determination of the diameters of LDL and HDL from whole plasma. The matching of the concentrations at the interface of the two linear gradients in this embodiment provides a continuous transition between the two linear gradients.
In a further embodiment of the present invention, two or more gradient makers, each with two containers A and B, can be used to simultaneously make two or more gradient gels in two or more gel chambers. Therefore, practicing the present invention is economic and efficient, in addition to the advantages of making better continuous gradient gels.
In an additional further embodiment of the present invention, multiple linear gradients can be stacked on top of another to allow optimal separation of the macromolecules of interest. For example, the present invention can be practiced to make a linear gradient gel with three segments. Similarly, gels having other concentration gradients can also be made.
Other objects, advantages and uses for the present invention will be more clearly understood by reference to the remainder of this document.